Differentiation of functional osteoclasts from hESC and hiPSCs. Representative micrographs of multinucleated osteoclasts derived from H1-derived (A-B) and MSC-iPS1–derived (C-D) hematopoietic precursors. Cells from day 20 cultures were seeded onto dentine slices in the presence of M-CSF and RANKL, and osteoclasts were stained 2 weeks later for TRAP activity (↑). Clear multinucleation is evident (B,D) and prominent resorption trails (*) are associated with all osteoclasts as viewed under reflected light. Resorption lacunae created by H1-derived (E,F,I) and MSC-iPS1–derived (G-H) osteoclasts were identified after removal of cells and staining with toluidine blue or by scanning electron microscopy (I). Cells cultured in the absence of RANKL did not differentiate into TRAP-positive cells, and resorption pits were never observed (J). Images were acquired with a Leica MZ FLIII stereomicroscope using a DFC300 FX camera. Auto adjustments were performed with Adobe Photoshop software. Original magnifications: ×4 (E); ×10 (A,C,G,J); ×20 (F,H); and ×40 (B,D).